Epithelial Na(+) Channel Inhibition Research Articles

#1825 Treating hyperphosphatemia in CKD via targeting Npt2a in the kidney

Abstract Background and Aims Targeting the renal sodium phosphate cotransporter 2a (Npt2a) offers a novel strategy for treating hyperphosphatemia in chronic kidney disease (CKD). In patients with and without reduced kidney function, hyperphosphatemia is associated with cardiovascular complications and currently treatment options are limited. Method To study the role of a novel Npt2a inhibitor (PF-06869206) we used in vitro (Opossum kidney [OK] cells) and in vivo approaches (C57Bl/6 and Npt2a knockout [Npt2a−/−] mice). As models of CKD, 5/6 nephrectomy (5/6 Nx) as well as novel Alport/Npt2a knockout mice (Alport/Npt2a−/−) were studied. Results OK cell experiments showed that the Npt2a inhibitor caused dose-dependent reductions of Na+-dependent Pi uptake (IC50: ∼1.4 μmol/L) and Michaelis-Menten kinetics identified a ∼2.4-fold higher Km for Pi in response to Npt2a inhibition with no significant change in apparent Vmax, indicating a competitive mode of action. In vivo, Npt2a inhibition enhanced urinary excretions of Pi, Ca2+, and Na+ dose-dependently, which is recapitulated in animal models with reduced kidney function (5/6 Nx and Alport mice). Reductions in plasma Pi occur within 30 minutes and reach a maximum (40-50%) after 2 hours, this coincided with ∼50% reductions in PTH levels. No effects on FGF23 were observed in this time frame. In C57Bl/6 mice qualitative immunohistochemistry after 24 hours showed reductions in Npt2a labelling in apical microvilli compared to vehicle treatment. Providing proof of concept, Npt2a−/− and Alport/Npt2a−/− mice confirmed specificity of the drug, effects on urinary and plasma Pi were absent. Further electrophysiological studies in acutely isolated connecting tubule/cortical collecting duct showed that open probability of the epithelial Na+ channel (ENaC) was reduced in response to Npt2a inhibition, suggesting that the natriuretic effect is mediated, at least in part, by inhibition of ENaC. Conclusion Npt2a inhibition offers a promising therapeutic approach for treating hyperphosphatemia and possibly offers the opportunity of reducing cardiovascular complications in CKD.

Reverse Phenotypes of Patients with Genetically Confirmed Liddle Syndrome.

Liddle syndrome was initially characterized by hypertension, hypokalemia, metabolic alkalosis, and suppressed plasma renin and aldosterone, resulting from gain-of-function variants in the epithelial Na + channel (ENaC). Efficient treatment with ENaC inhibitors is available, but the phenotypic spectrum of genetically confirmed Liddle syndrome is unknown, and some patients may remain undiagnosed and at risk of inefficient treatment. In this study, we used a reverse phenotyping approach to investigate the Liddle syndrome phenotypic spectrum and genotype-phenotype correlations. Pubmed, Embase, Scopus, and the Human Gene Mutation Database were searched for articles reporting Liddle syndrome variants. The genetic variants were systematically classified to identify patients with genetically confirmed Liddle syndrome. We identified 62 articles describing 45 unique variants within 86 Liddle syndrome families, and phenotypic data were pooled for 268 patients with confirmed Liddle syndrome. The Liddle syndrome variants localized to exon 13 of SCNN1B and SCNN1G , disrupting the PPPxY motif critical for downregulating ENaC activity. Hypertension sensitive to ENaC inhibition was present in 97% of adults carrying Liddle syndrome variants while hypokalemia, metabolic alkalosis, and plasma renin and aldosterone suppression showed incomplete penetrance. In addition, 95% and 55% of patients had a family history of hypertension or cerebrovascular events, respectively. The genotype had minor phenotypic effects; however, probands compared with relatives showed significant phenotypic discrepancies consistent with selection bias for initial genetic screening. Patients with genetically confirmed Liddle syndrome displayed a phenotypic spectrum, with ENaC-sensitive hypertension and family history of hypertension being the most common features. The phenotype seemed independent of the specific gene or variant type involved.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Epithelial sodium channels in macrophage migration and polarization: role of proinflammatory cytokines TNFα and IFNγ.

Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na+ channels (ENaCs) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH2-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.

Deficient regulation of the epithelial Na+ channel (ENaC) activity and impaired urinary conservation of Na+ in Epac1 ‐/‐ and Epac2 ‐/‐ mice

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1‐/‐ and Epac2‐/‐ mice exhibit a delayed anti‐natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split‐opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the knockout mice fed with Na+ deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker, ESI‐05 and Epac1&2 blocker, ESI‐09 decreased ENaC activity in EpacWT mice kept on Na+ deficient diet but not on the regular diet. ESI‐09 injections led to natriuresis in EpacWT mice on Na+ deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate non‐redundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC over‐activation.

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor

The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Epac1-/- and Epac2-/- mice exhibit deficient epithelial Na+ channel regulation and impaired urinary Na+ conservation.

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1–/– and Epac2–/– mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.

Benzamil-mediated urine alkalization is caused by the inhibition of H+-K+-ATPases.

Epithelial Na+ channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H+ secretion via renal H+-K+-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K+ intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH4+ and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K+ diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K+ diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α1-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function.NEW & NOTEWORTHY Bolus application of epithelial Na+ channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H+-K+ pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.

LPS-induced epithelial barrier disruption via hyperactivation of CACC and ENaC.

Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases, including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated its underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that the apical application of LPS evoked short-circuit current (Isc) through the activation of CACC and ENaC. Although LPS disrupted epithelial barrier integrity, this was restored with the inhibition of CACC and ENaC, indicating the role of CACC and ENaC in the regulation of paracellular pathways. We confirmed that LPS, CACC, or ENaC activation evoked apical membrane depolarization. The exposure to a high-K+ buffer increased paracellular permeability. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, although LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections, this vulnerability was prevented by inhibiting CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and the subsequent tyrosine phosphorylation of ZO-1.

Sex differences in solute transport along the nephrons: effects of Na+ transport inhibition.

Each day, ~1.7 kg of NaCl and 180 liters of water are reabsorbed by nephron segments in humans, with urinary excretion fine tuned to meet homeostatic requirements. These tasks are coordinated by a spectrum of renal Na+ transporters and channels. The goal of the present study was to investigate the extent to which inhibitors of transepithelial Na+ transport (TNa) along the nephron alter urinary solute excretion and how those effects may vary between male and female subjects. To accomplish that goal, we developed sex-specific multinephron models that represent detailed transcellular and paracellular transport processes along the nephrons of male and female rat kidneys. We simulated inhibition of Na+/H+ exchanger 3 (NHE3), bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2), Na+-Cl- cotransporter (NCC), and amiloride-sensitive epithelial Na+ channel (ENaC). NHE3 inhibition simulations predicted a substantially reduced proximal tubule TNa, and NKCC2 inhibition substantially reduced thick ascending limb TNa. Both gave rise to diuresis, natriuresis, and kaliuresis, with those effects stronger in female rats. While NCC inhibition was predicted to have only minor impact on renal TNa, it nonetheless had a notable effect of enhancing excretion of Na+, K+, and Cl-, particularly in female rats. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na+ (increased) and K+ (decreased) and to have only a minor impact on whole kidney TNa. Unlike inhibition of other transporters, ENaC inhibition induced stronger natriuresis and diuresis in male rats than female rats. Overall, model predictions agreed well with measured changes in Na+ and K+ excretion in response to diuretics and Na+ transporter mutations.

Cisplatin Decreases ENaC Activity Contributing to Renal Salt Wasting Syndrome.

Cisplatin (CDDP) is an important anticancer drug. A common side effect of CDDP is renal salt and water-wasting syndrome (RSWS). The origin of RSWS is obscure. Emerging evidence, though, suggests that broad inhibition of sodium transport proteins by CDDP may result in decreases in tubular reabsorption, causing increases in sodium and water excretion. In this sense, CDDP would be acting like a diuretic. The effect of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter fine-tuning renal Na+ excretion, is unknown. We test here whether CDDP affects ENaC to promote renal salt and water excretion. The effects of CDDP and benzamil (BZM), a blocker of ENaC, on excretion of a sodium load were quantified. Similar to BZM, CDDP facilitated renal Na+ excretion. To directly quantify the effects on ENaC, principal cells in split-open tubules were patch clamped. CDDP, at doses comparable to those used for chemotherapy (1.5 µM), significantly decreased ENaC activity in native tubules. To further elaborate on this mechanism, the dose-dependent effects of CDDP on mouse ENaC (mENaC) heterologously expressed in Chinese Hamster Ovary (CHO) cells were tested using patch clamping. As in native tubules, CDDP significantly decreased the activity of mENaC expressed in CHO cells. Dose–response curves and competition with amiloride identified CDDP as a weak inhibitor of ENaC (apparent IC50 = 1 µM) that competes with amiloride for inhibition of the channel, weakening the inhibitory actions of the latter. Such observations are consistent with CDDP being a partial modulator of ENaC, which possibly has a binding site that overlaps with that of amiloride. These findings are consistent with inhibition of ENaC by CDDP contributing to the RSWS caused by this important chemotherapy drug.

ENaC inhibition in cystic fibrosis: potential role in the new era of CFTR modulator therapies.

ENaC inhibition with BI 1265162 is a promising strategy to optimise outcomes in patients with CF either eligible, or ineligible, for CFTR modulator therapy. Phase II clinical trials of BI 1265162 must now show this translates into clinical benefit. https://bit.ly/2OQ1IUI

AMPK phosphorylation of the β1Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β1 β1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional β1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates β1Pix in vitro. Among several AMPK phosphorylation sites on β1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type β1Pix, overexpression of a phosphorylation-deficient β1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to β1Pix is critical for the formation of a β1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β1Pix at Ser71, which promotes 14-3-3 interactions with β1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

Renal Na+ excretion consequent to pharmacogenetic activation of Gq-DREADD in principal cells.

Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.

Lack of urea transporters, UT-A1 and UT-A3, increases nitric oxide accumulation to dampen medullary sodium reabsorption through ENaC.

Although the role of urea in urine concentration is known, the effect of urea handling by the urea transporters (UTs), UT-A1 and UT-A3, on sodium balance remains elusive. Serum and urinary sodium concentration is similar between wild-type mice (WT) and UT-A3 null (UT-A3 KO) mice; however, mice lacking both UT-A1 and UT-A3 (UT-A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters is unchanged among all three genotypes. WT, UT-A3 KO, and UT-A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT-A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration, indicating that baseline epithelial Na+ channel (ENaC) activity is impaired. UT-A1/A3 KO have more ENaC at the apical membrane than WT mice, and single-channel analysis of ENaC in split-open inner medullary collecting duct (IMCD) isolated in saline shows that ENaC channel density and open probability is higher in UT-A1/A3 KO than WT. UT-A1/A3 KO excrete more urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and inner medullary nitric oxide synthase 1 mRNA expression is ~40-fold higher than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split-open IMCD with the NO donor PAPA NONOate [1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)], and ENaC activity was almost abolished in UT-A1/A3 KO. In summary, loss of both UT-A1 and UT-A3 (but not UT-A3 alone) causes elevated medullary NO production and salt wasting. NO inhibition of ENaC, despite elevated apical accumulation of ENaC in UT-A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT-A1/A3 KO mice.

β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epithelial Na+ channel differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice.

A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.

Bumetanide increases Cl--dependent short-circuit current in late distal colon: Evidence for the presence of active electrogenic Cl- absorption

Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl-. The present studies employing Ussing chamber technique identified two opposing short-circuit current (Isc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl--secretory Isc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated Isc in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na+-K+-2Cl- cotransporter (NKCC) in crypt cells and basolateral K+-Cl- cotransporter (KCC) in surface epithelium, we proposed this stimulatory Isc could represent a KCC-mediated Cl- absorptive current. In support of this hypothesis, ion substitution experiments established Cl- dependency of this absorptive Isc and transport inhibitor studies demonstrated the involvement of an apical Cl- conductance. Current distribution and RNA sequencing analyses revealed that this Cl- absorptive Isc is closely associated with epithelial Na+ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 μM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl- absorptive Isc senses dietary Cl- content; thus when dietary Cl- was low, Cl- absorptive Isc was up-regulated. In contrast, when dietary Cl- was increased, Cl- absorptive Isc was down-regulated. We conclude that an active Cl- extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.

ENaC and ROMK activity are inhibited in the DCT2/CNT of TgWnk4PHAII mice.

Mice transgenic for genomic segments harboring PHAII (pseudohypoaldosteronism type II) mutant Wnk4 (with-No-Lysine kinase 4) (TgWnk4PHAII) have hyperkalemia which is currently believed to be the result of high activity of Na-Cl cotransporter (NCC). This leads to decreasing Na+ delivery to the distal nephron segment including late distal convoluted tubule (DCT) and connecting tubule (CNT). Since epithelial Na+ channel (ENaC) and renal outer medullary K+ channel (ROMK or Kir4.1) are expressed in the late DCT and play an important role in mediating K+ secretion, the aim of the present study is to test whether ROMK and ENaC activity in the DCT/CNT are also compromised in the mice expressing PHAII mutant Wnk4. Western blot analysis shows that the expression of βENaC and γENaC subunits but not αENaC subunit was lower in TgWnk4PHAII mice than that in wild-type (WT) and TgWnk4WT mice. Patch-clamp experiments detected amiloride-sensitive Na+ currents and TPNQ-sensitive K+ currents in DCT2/CNT, suggesting the activity of ENaC and ROMK. However, both Na+ and ROMK currents in DCT2/CNT of TgWnk4PHAII mice were significantly smaller than those in WT and TgWnk4WT mice. In contrast, the basolateral K+ currents in the DCT were similar among three groups, despite higher NCC expression in TgWnk4PHAII mice than those of WT and TgWnk4WTmice. An increase in dietary K+ intake significantly increased both ENaC and ROMK currents in the DCT2/CNT of all three groups. However, high-K+ (HK) intake-induced stimulation of Na+ and K+ currents was smaller in TgWnk4PHAII mice than those in WT and TgWnk4WT mice. We conclude that ENaC and ROMK channel activity in DCT2/CNT are inhibited in TgWnk4PHAII mice and that Wnk4PHAII-induced inhibition of ENaC and ROMK may contribute to the suppression of K+ secretion in the DCT2/CNT in addition to increased NCC activity.

Caffeine intake antagonizes salt sensitive hypertension through improvement of renal sodium handling.

High salt intake is a major risk factor for hypertension. Although acute caffeine intake produces moderate diuresis and natriuresis, caffeine increases the blood pressure (BP) through activating sympathetic activity. However, the long-term effects of caffeine on urinary sodium excretion and blood pressure are rarely investigated. Here, we investigated whether chronic caffeine administration antagonizes salt sensitive hypertension by promoting urinary sodium excretion. Dahl salt-sensitive (Dahl-S) rats were fed with high salt diet with or without 0.1% caffeine in drinking water for 15 days. The BP, heart rate and locomotor activity of rats was analyzed and urinary sodium excretion was determined. The renal epithelial Na+ channel (ENaC) expression and function were measured by in vivo and in vitro experiments. Chronic consumption of caffeine attenuates hypertension induced by high salt without affecting sympathetic nerve activity in Dahl-S rats. The renal α-ENaC expression and ENaC activity of rats decreased after chronic caffeine administration. Caffeine increased phosphorylation of AMPK and decrease α-ENaC expression in cortical collecting duct cells. Inhibiting AMPK abolished the effect of caffeine on α-ENaC. Chronic caffeine intake prevented the development of salt-sensitive hypertension through promoting urinary sodium excretion, which was associated with activation of renal AMPK and inhibition of renal tubular ENaC.